Microglia in the Brain Tumor Microenvironment.

Microglia are the brain resident phagocytes that act as the primary form of the immune defense in the central nervous system. These cells originate from primitive macrophages that arise from the yolk sac. Advances in imaging and single-cell RNA-seq technologies provided new insights into the complexity of microglia biology.Microglia play an essential role in the brain development and maintenance of brain homeostasis. They are also crucial in injury repair in the central nervous system. The tumor microenvironment is complex and includes neoplastic cells as well as varieties of host and infiltrating immune cells. Microglia are part of the glioma microenvironment and play a critical part in initiating and maintaining tumor growth and spread. Microglia can also act as effector cells in treatments against gliomas. In this chapter, we summarize the current knowledge of how and where microglia are generated. We also discuss their functions during brain development, injury repair, and homeostasis. Moreover, we discuss the role of microglia in the tumor microenvironment of gliomas and highlight their therapeutic implications.

Potential role for snoRNAs in PKR activation during metabolic stress.

Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKRWT) or PKR with a point mutation in each dsRNA-binding motif (PKRRM). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKRWT samples after PA treatment, but not in the PKRRM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5'-triphosphate enhanced PKR activity compared with the activity with a 5'-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.

Genome-wide profiling of the C. elegans dsRNAome.

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3' UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.

Parasitological evaluation of Ro 15-9268, a 9-acridanone-hydrazone derivative against Schistosoma mansoni in mice, and observations on changes in serum enzyme levels.

Schistosoma mansoni is one of the major causes of schistosomiasis prevalent in tropical and subtropical areas, especially in poor communities. It is estimated that at least 90% of those requiring treatment for schistosomiasis live in Africa. The primary control strategy employed for schistosomiasis is mass drug administration (MDA).The aim is to reduce disease through treatments with a single lower dose of Ro 15-9268 as a new antischistosomal drug. In the present search, the efficacy of Ro 15-9268 was studied in mice using a dose of 12.5 mg/kg of body weight (b.wt.) against an Egyptian strain of S. mansoni. This was carried out at 2 days and 3, 4, and 6 weeks post-cercarial exposure of mice. The criteria used were the worm load, oogram pattern and number of ova in the liver and intestine, hepatic enzyme activity, and liver histopathology. The tested agent has led to a significant reduction in worm burden (89.80%) in liver and portomesenteric veins concurrent with a hepatic shift at the second week posttreatment followed by a complete disappearance of worms, 4 weeks postmedication. The oogram of infected animals treated revealed an increased number of dead ova 2 days posttreatment and complete absence of immature and mature ova 2 weeks later. The hepatic and intestinal egg counts significantly declined by about 96 and 98%, respectively, 6 weeks after treatment, and the fecal egg count completely disappeared from stool 4 weeks after medication. The hepatic histopathological changes were improved, ova were markedly degenerated, and worms showed fragmentation and degeneration after drug administration. In conclusion, when Ro 15-9268 was administered to mice infected with the Egyptian strain of S. mansoni, at a low dose level (12.5 mg/kg b.wt.), encouraging results were obtained. The drug showed high efficacy against schistosomal worms as well as histopathological inflammatory changes.

Morphological and ultrastructural investigation of the posterior atlanto-occipital membrane: Comparing children with Chiari malformation type I and controls.

INTRODUCTION: The fibrous posterior atlanto-occipital membrane (PAOM) at the craniocervical junction is typically removed during decompression surgery for Chiari malformation type I (CM-I); however, its importance and ultrastructural architecture have not been investigated in children. We hypothesized that there are structural differences in the PAOM of patients with CM-I and those without. METHODS: In this prospective study, blinded pathological analysis was performed on PAOM specimens from children who had surgery for CM-I and children who had surgery for posterior fossa tumors (controls). Clinical and radiographic data were collected. Statistical analysis included comparisons between the CM-I and control cohorts and correlations with imaging measures. RESULTS: A total of 35 children (mean age at surgery 10.7 years; 94.3% white) with viable specimens for evaluation were enrolled: 24 with CM-I and 11 controls. There were no statistical demographic differences between the two cohorts. Four children had a family history of CM-I and five had a syndromic condition. The cohorts had similar measurements of tonsillar descent, syringomyelia, basion to C2, and condylar-to-C2 vertical axis (all p>0.05). The clival-axial angle was lower in patients with CM-I (138.1 vs. 149.3 degrees, p = 0.016). Morphologically, the PAOM demonstrated statistically higher proportions of disorganized architecture in patients with CM-I (75.0% vs. 36.4%, p = 0.012). There were no differences in PAOM fat, elastin, or collagen percentages overall and no differences in imaging or ultrastructural findings between male and female patients. Posterior fossa volume was lower in children with CM-I (163,234 mm3 vs. 218,305 mm3, p<0.001), a difference that persisted after normalizing for patient height (129.9 vs. 160.9, p = 0.028). CONCLUSIONS: In patients with CM-I, the PAOM demonstrates disorganized architecture compared with that of control patients. This likely represents an anatomic adaptation in the presence of CM-I rather than a pathologic contribution.

In Vitro Macrophage-Mediated Phagocytosis Assay of Brain Tumors.

Tumor-associated macrophages (TAMs) have recently emerged as potentially crucial therapeutic targets for cancer. Thus, the development of macrophage-mediated phagocytosis assays is vital for preclinical drug screening of different tumor cells. This assay can be used to evaluate the effect of anti-cancer therapy, such as immunotherapy, radiotherapy, and chemotherapy, on different tumor cells. Here, we describe the in-vitro phagocytosis assay in detail. As an example of immunotherapy treatment, we used a monoclonal antibody to block an anti-phagocytic signal (CD47) to evaluate the assay using human brain tumor cells and monocyte-derived macrophages. We also demonstrated that this assay can be used to evaluate the effect of different irradiation doses on the phagocytosis of brain tumor cells. This functional assay is fast, accurate, and highly reproducible. Furthermore, the results successfully demonstrate that anti-CD47 antibodies and irradiation can enhance the macrophage-mediated phagocytosis of brain tumors.

Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP.

H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5-guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site.

Management of brain metastases with stereotactic radiosurgery alone versus whole brain irradiation alone versus both.

INTRODUCTION: This prospective randomized study aimed to evaluate the role of WBRT + SRS compared to SRS alone and to WBRT alone in improvement of overall survival, brain local control and neurologic manifestations. PATIENTS AND METHODS: The trial included 60 patients with 1 to 3 brain metastases treated at the Radiotherapy Department, National Cancer Institute. 21 patients received WBRT + SRS, 18 patients received SRS alone and 21 patients received WBRT alone. RESULTS: Median local control was significantly better for WBRT + SRS compared to SRS alone & WBRT alone (10 vs 6 vs 5 months, respectively, P = 0.04). There was non significant survival benefit for WBRT + SRS compared to SRS alone & WBRT alone. Survival was significantly better for patients with controlled primary tumor who received WBRT + SRS compared to SRS alone & WBRT alone (median survival was 12 vs 5.5 vs 8 months, respectively. P = 0.027). Regardless of the treatment group, median survival and median local control were highly significantly better for single brain site involvement compared to multiple brain sites involvement (P = 0.003 & P = 0.001, respectively), and median brain local control was significantly better for single lesion compared to multiple lesions (P = 0.05). CONCLUSIONS: WBRT + SRS is an effective, safe tool in treatment of patients with 1 to 3 brain metastses improving the brain local control, but further studies with larger number of patients is recommended.

Simplified local anesthesia technique for external dacryocystorhinostomy without nasal packing: a new technique and pilot study outcome.

BACKGROUND: The purpose of this paper is to describe a simplified local anesthesia technique for external dacryocystorhinostomy (EXT-DCR). METHODS: In this pilot, retrospective, noncomparative, interventional case series, 448 patients (480 eyes) underwent EXT-DCR using a simplified local anesthesia technique. Nasal mucosal anesthesia was achieved using combined application of 6 mL of oxymetazoline 0.025% nasal spray and lidocaine 1% in the same spray bottle, without any packing of the nose with either pledgets or ribbon gauze. Local infiltration anesthesia consisted of subcutaneous injection of a 7 mL mixture of 2% lidocaine with 1:100,000 epinephrine injected on the flat side of the nose beneath the incision site, in addition to a second medial peribulbar injection (3 mL, 2% lidocaine without epinephrine). RESULTS: Successful unilateral or bilateral EXT-DCR was achieved in 432/448 patients (96.4%). Four patients could not tolerate the procedure under local anesthesia and were converted to general anesthesia. Four patients required additional local anesthetic injections because of intolerable pain. Heavy sedation was essential in eight uncooperative patients because surgical manipulation was impossible. The remaining patients tolerated the procedure well. The intraoperative bleeding rate was very low except in one patient. Mean operative time was 16 minutes. Severe postoperative epistaxis was observed in four patients. Temporary anosmia developed in one patient. CONCLUSION: Our simplified local anesthesia approach of EXT-DCR is convenient for the patient because it avoids unnecessary nasal packing. It is also safe and effective, as evidenced by the high rate of successful completion of the procedure without conversion to general anesthesia or the need for supplemental local anesthesia.

Crystal structure of a Cbf5-Nop10-Gar1 complex and implications in RNA-guided pseudouridylation and dyskeratosis congenita.

H/ACA RNA-protein complexes, comprised of four proteins and an H/ACA guide RNA, modify ribosomal and small nuclear RNAs. The H/ACA proteins are also essential components of telomerase in mammals. Cbf5 is the H/ACA protein that catalyzes isomerization of uridine to pseudouridine in target RNAs. Mutations in human Cbf5 (dyskerin) lead to dyskeratosis congenita. Here, we describe the 2.1 A crystal structure of a specific complex of three archaeal H/ACA proteins, Cbf5, Nop10, and Gar1. Cbf5 displays structural properties that are unique among known pseudouridine synthases and are consistent with its distinct function in RNA-guided pseudouridylation. We also describe the previously unknown structures of both Nop10 and Gar1 and the structural basis for their essential roles in pseudouridylation. By using information from related structures, we have modeled the entire ribonucleoprotein complex including both guide and substrate RNAs. We have also identified a dyskeratosis congenita mutation cluster site within a modeled dyskerin structure.

RNA-guided RNA modification: functional organization of the archaeal H/ACA RNP.

In eukaryotes and archaea, uridines in various RNAs are converted to pseudouridines by RNA-guided RNA modification complexes termed H/ACA RNPs. Guide RNAs within the complexes base-pair with target RNAs to direct modification of specific ribonucleotides. Cbf5, a protein component of the complex, likely catalyzes the modification. However, little is known about the organization of H/ACA RNPs and the roles of the multiple proteins thought to comprise the complexes. We have reconstituted functional archaeal H/ACA RNPs from recombinant components, defined the components necessary and sufficient for function, and determined the direct RNA-protein and protein-protein interactions that occur between the components. The results provide substantial insight into the functional organization of this RNP. The functional complex requires a guide RNA and each of four proteins: Cbf5, Gar1, L7Ae, and Nop10. Two proteins interact directly with the guide RNA: L7Ae and Cbf5. L7Ae does not interact with other H/ACA RNP proteins in the absence of the RNA. We have defined two novel functions for Cbf5. Cbf5 is the protein that specifically recognizes and binds H/ACA guide RNAs. In addition, Cbf5 recruits the two other essential proteins, Gar1 and Nop10, to the pseudouridylation guide complex.